The present application relates to a fluorescence intensity compensation method and a fluorescence intensity calculation device. More particularly, the present application relates to a fluorescence intensity compensation method and the like, to accurately calculate the intensity of fluorescence emitted from each of multiple fluorochromes multiply-labeling microparticles.
There are devices according to the related art which measure properties of microparticles (e.g., flow cytometers) which measure the properties of microparticles, by marking microparticles such as cells or the like using fluorochromes, exciting these by laser beam irradiation, and measuring fluorescence intensities and patterns emitted from these fluorochromes. In recent years, multicolor measurement has come to be performed to analyze the properties cells and the like in greater detail, in which microparticles are marked using multiple fluorochromes, and light emitted from each fluorochromes is measured with multiple photodetectors having different input wavelength bands (PD, PMT, etc.). With multicolor measurement, detection of fluorescence is performed by selecting optical filters at the photodetector side in accordance with the fluorescence wavelength of the fluorochromes used.
On the other hand, currently-used fluorochromes (e.g., FITC, PE (phycoerythrin), APC (allophycocyanin), etc.) have mutually-overlapping wavelength bands in the fluorescence spectrum. Accordingly, in the event of combining these fluorochromes to perform multicolor measurement, fluorescence from unintended fluorochromes may leak into the photodetectors even if the fluorescence emitted from the fluorochromes is separated into wavelength bands by optical filters. If leakage of fluorescence occurs, there will be discrepancy between the fluorescence intensity measured at the detectors and the true fluorescence intensity from the intended fluorochromes, leading to measurement error.
Fluorescence compensation is performed in which fluorescence intensity equivalent to the leakage is subtracted from the fluorescence intensity measured at the photodetector is performed in order to compensate for the measurement error. Fluorescence compensation involves applying electrical or mathematical correction to pulses so that the fluorescence intensity measured at the photodetector is the true fluorescence intensity from the intended fluorochrome.
A technique is being used as a method for mathematically performing fluorescence compensation in which the true fluorescence intensity from the intended fluorochromes is calculated, by representing the fluorescence intensities (detected values) measured at the photodetectors are represented as vectors, and an inverse matrix of a preset leakage matrix is applied to these vectors (see FIGS. 12 and 13, and Japanese Unexamined Patent Application Publication No. 2003-83894). This leakage matrix is created by analyzing fluorescence wavelength distribution of microparticles single-labeled individually with the fluorochromes, with the fluorescence wavelengths of the fluorochromes arrayed as column vectors. An inverse matrix of a leakage matrix is also called a “compensation matrix”. FIGS. 12 and 13 show an example of performing five-color measurement using five types of fluorochromes (FITC, PE, ECD, PC5, PC7) and five photodetectors.